Project Summary This is a proposal by investigators in neurophysiology, neurodevelopment, immunology, microbiology, and neurodegeneration for a Shared Instrumentation Grant to purchase an Olympus Fluoview Multiphoton Excitation Resonance Scanning (FVMPE-RS) Microscope equipped with two Spectra-Physics Ti:Sapphire Ultrafast Lasers. The Olympus FVMPE-RS is an innovative, state-of-the-art two-photon (2P) laser-scanning microscope that allows for simultaneous fluorophore excitation and imaging at two different wavelengths and is specially designed for in vivo imaging applications in neuroscience. By permitting the concurrent excitation of two genetically encoded calcium sensors with discernible emission spectra expressed independently in different neuronal subtypes, it is possible to assess the activation and interaction of different neuronal networks in behaving animals. The specially designed gantry system allows for 355 mm of clearance between the microscope objective and the baseplate, enabling users to install customized behavioral equipment for especially complex behavioral assays to be performed concurrently with in vivo imaging. The same equipment will also allow for simultaneous 2P imaging and 2P optogenetic stimulation, enabling the investigators not only to record from neuronal networks but also to manipulate their activity in real time, with single-cell precision. The requested instrument would enable Weill Cornell investigators to conduct multidisciplinary experiments spanning neurophysiology, developmental neurobiology, immunology, and microbiology.